1. Field of the Invention
There is an expanding interest in the ability to determine or monitor small amounts of organic antigenic or haptenic materials. Frequently, the concentrations of interest are micromolar or less. Various techniques have been developed which are capable of isolating and detecting a specific compound, despite the presence of myriad other compounds of similar and different structure.
One group of techniques referred to as competitive protein binding assays or immunoassays depend for their specificity on the use of a receptor, normally an antibody, which is specific for a compound of a particular spatial and polar organization. For these assays it is normally necessary to produce antibodies by injecting antigens or hapten conjugated antigens into a vertebrate to induce the formation of antibodies which may then be harvested. The bleeds which are obtained can be usually purified to separate a globulin concentrate from other proteinaceous materials. To further purify the globulin concentrate to separate the antibodies of interest from other globulins is only difficultly achieved.
While affinity chromatography will provide for some concentration of the desired antibodies, the procedure is normally slow and frequently results in substantial loss of the desired antibodies as well as reduction in the binding constant. That is, those antibodies in the composition which have the strongest binding, frequently cannot be removed from the column. Therefore, most methods have avoided labeling antibodies, since either the antibodies had to be purified to concentrate the antibodies of interest or a large amount of label was introduced associated with proteins which were not involved in the assay. This normally results in a large background signal which inevitably reduces the sensitivity of the assay, unless a physical separation of the extraneous label is incorporated into the procedure.
The alternative has been to label ligand. While labeling of ligand is feasible where the ligand is a simple hapten or the antigens are available in substantially pure form, in those situations where the antigen is only difficultly purifiable, exists in only small amounts, or is labile, labeling of antigens is not feasible for a commercial process.
An assay is therefore desirable which avoids the problems of purification and isolation of both the antibodies of interest and the ligand of interest. In addition, the assay should provide for minimizing introduction of label into the assay medium which produces a signal which interferes with or is additive with the signal which is measured.
2. Brief Description of the Prior Art
Radioimmunoassay is described in two articles by Murphy, J. Clin. Endocr. 27, 973 (1967); ibid 28, 343 (1968). U.S. Pat. No. 3,817,837 teaches a homogeneous enzyme immunoassay. U.S. Pat. Nos. 3,654,090, 3,791,932, 3,850,752 and 3,839,153 teach hetereogeneous enzyme immunoassays. In the agenda for the Ninth Annual Symposium on Advanced Analytical Concepts for the Clinical Laboratory, to be held March 17 and 18, 1977 at the Oakridge National Laboratory, a paper entitled "Phospholipase C-Labeled Antihuman IgG: Inhibition of Enzyme Activity by human IgG," to be presented by R. Wei and S. Riebe is reported. U.S. Pat. Nos. 3,935,074 and 3,998,943 disclose immunoassay techniques involving steric inhibition between two different receptors for different epitopic sites. U.S. Pat. No. 3,996,345 teaches the use of a common receptor, a portion of which is bound to a fluorescer and the remaining portion bound to quencher, whereby the presence of ligand brings the receptors together so as to allow for quenching of fluorescence. Carrico, et al, Anal. Biochem. 72 271 (1976) and Schroder, et al, ibid 72 283 (1976) teach competitive protein binding assays where a label is bonded to a hapten with the label being subject to enzymatic transformation to produce a signal. Antibody bound to the hapten inhibits the approach of enzyme to the label.